Claims for Patent: 5,270,181

Summary for Patent: 5,270,181

Title:	Peptide and protein fusions to thioredoxin and thioredoxin-like molecules
Abstract:	This invention provides a fusion molecule comprising a DNA sequence encoding a thioredoxin-like protein fused to the DNA sequence encoding a selected heterologous peptide or protein. The peptide or protein may be fused to the amino terminus of the thioredoxin-like molecule, the carboxyl terminus of the thioredoxin-like molecule, or within the thioredoxin-like molecule, for example at the active-site loop of said molecule. Expression of this fusion molecule under the control of a regulatory sequence capable of directing its expression in a desired host cell, produces high levels of stable and soluble fusion protein. The fusion protein, located in the bacterial cytoplasm, may be selectively released from the cell by osmotic shock or freeze/thaw procedures. It may be optionally cleaved to liberate the soluble, correctly folded heterologous protein from the thioredoxin-like portion.
Inventor(s):	McCoy; John (Reading, MA), LaVallie; Edward R. (Tewksbury, MA)
Assignee:	Genetics Institute, Inc. (Cambridge, MA)
Application Number:	07/745,382
Patent Claims:	1. Fusion DNA having a formula selected from the group consisting of R1-R2, R2-R1, R1-L-R2 and R2-L-R1 wherein, R1 is thioredoxin-like DNA; R2 is eukaryotic DNA fused in frame and encoding a selected, desired protein; and L is linker DNA fused in frame and encoding a proteolytic cleavage site between the polypeptides encoded by R1 and R2 and preventing steric hindrance therebetween, said thioredoxin-like DNA being characterized by encoding a polypeptide that (1) has a three-dimensional structure substantially similar to that of E. coli thioredoxin and (2) contains an active site loop functionally and structurally equivalent to the double cysteine containing active site loop of E. coli thioredoxin. 2. Fusion DNA comprising (1) thioredoxin-like DNA, said thioredoxin-like DNA characterized by encoding a polypeptide that (a) has a three-dimensional structure substantially similar to that of E. coli thioredoxin and (b) contains an active site loop functionally and structurally equivalent to the double cysteine containing active site loop of E. coli thioredoxin; and (2) DNA encoding a selected, desired protein or peptide and fused in frame within said active site of said thioredoxin-like DNA. 3. The fusion DNA of claim 2 additionally comprising a linker DNA encoding a proteolytic cleavage site and fused in frame between said thioredoxin-like DNA and said selected, desired DNA. 4. The fusion DNA of claim 1 wherein R1 is selected from the group consisting of a DNA encoding thioredoxin, glutaredoxin, and the thioredoxin-like domains of protein disulfide isomerase, form-1 phosphoinositide-specific phospholipase C, and ERp72. 5. The fusion DNA of claim 3 wherein R1 is selected from the group consisting of a DNA encoding thioredoxin, glutaredoxin, and the thioredoxin-like domains of protein disulfide isomerase, form-1 phosphoinositide-specific phospholipase C, and ERp72. 6. The fusion DNA of claim 1 wherein R2 is selected from the group consisting of a DNA encoding IL-11, IL-6, M-CSF, MIP-1.alpha. and BMP-2. 7. Fusion DNA having a formula selected from the group consisting of R1-R2, R2-R1, R1-L-R2 and R2-L-R1 wherein, R1 is DNA encoding E. coli thioredoxin; R2 is eukaryotic DNA fused in frame and encoding a selected, desired protein; and L is linker DNA fused in frame and encoding a proteolytic cleavage site between the polypeptides encoded by R1 and R2 and preventing steric hindrance therebetween. 8. The fusion DNA of claim 7 wherein R2 is selected from the group consisting of a DNA encoding IL-11, IL-6, M-CSF, MIP-1.alpha. and BMP-2. 9. The fusion DNA of claim 7 wherein R2 is a DNA encoding IL-11. 10. Fusion DNA having a formula R1-L-R2, wherein R1 is DNA encoding E. coli thioredoxin; R2 is in frame DNA encoding IL-11 and L is in frame linker DNA encoding G-S-G-S-G-D-D-D-D-K (amino acids 110-119 of SEQ ID No. 14). 11. The fusion DNA of claim 10 wherein DNA encoding IL-11 comprises nucleotides 2599 through 3132 of FIG. 1. 12. An expression vector comprising a DNA of claim 1. 13. An expression vector comprising a DNA of claim 4. 14. An expression vector comprising a DNA of claim 5. 15. An expression vector comprising a DNA of claim 7. 16. An expression vector comprising a DNA of claim 8. 17. An expression vector comprising a DNA of claim 10. 18. A host cell containing an expression vector of claim 13. 19. A host cell containing an expression vector of claim 14. 20. A host cell containing an expression vector of claim 15. 21. A host cell containing an expression vector of claim 17. 22. The host cell of claims 18, 19, 20 or 21 wherein said host cell is a bacterial host cell. 23. The host cell of claim 22 wherein said bacterial host cell is an E. coli bacterial host cell. 24. The host cell of claims 18, 19, 20 or 21 wherein said host cell is a yeast host cell. 25. A method of producing a selected recombinant protein comprising culturing under suitable culture conditions promoting protein production a host cell containing a DNA of claims 4, 6, 7 or 10 and recovering said selected recombinant protein from the culture medium. 26. The method according to claim 25 wherein said recovering step comprises treating said host cells by osmotic shock to release said protein from the cell. 27. The method according to claim 25 wherein said recovering step comprises treating said host cells by freezing and thawing to release said protein from the cell. 28. The method of claim 25 wherein said DNA comprises the DNA of claim 8. 29. The method of claim 25 wherein said DNA comprises the DNA of claim 9. 30. The method of claim 25 wherein said DNA comprises the DNA of claim 11.

Details for Patent 5,270,181

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Wyeth Pharmaceuticals Inc.	NEUMEGA	oprelvekin	For Injection	103694	November 25, 1997	5,270,181	2011-02-06

International Patent Family for US Patent 5,270,181

Country	Patent Number	Estimated Expiration
World Intellectual Property Organization (WIPO)	9516044
World Intellectual Property Organization (WIPO)	9402502
World Intellectual Property Organization (WIPO)	9213955